A Diagnostic Chip for the Colorimetric Detection of Legionella pneumophila in Less than 3 h at the Point of Need.

Legionella pneumophila has been pinpointed by the World Health Organization as the highest health burden of all waterborne pathogens in the European Union and is responsible for many disease outbreaks around the globe. Today, standard analysis methods (based on bacteria culturing onto agar plates) need several days (~12) in specialized analytical laboratories to yield results, not allowing for timely actions to prevent outbreaks. Over the last decades, great efforts have been made to develop more efficient waterborne pathogen diagnostics and faster analysis methods, requiring further advancement of microfluidics and sensors for simple, rapid, accurate, inexpensive, real-time, and on-site methods. Herein, a lab-on-a-chip device integrating sample preparation by accommodating bacteria capture, lysis, and DNA isothermal amplification with fast (less than 3 h) and highly sensitive, colorimetric end-point detection of L. pneumophila in water samples is presented, for use at the point of need. The method is based on the selective capture of viable bacteria on on-chip-immobilized and -lyophilized antibodies, lysis, the loop-mediated amplification (LAMP) of DNA, and end-point detection by a color change, observable by the naked eye and semiquantified by computational image analysis. Competitive advantages are demonstrated, such as low reagent consumption, portability and disposability, color change, storage at RT, and compliance with current legislation.

Poly-L-histidine coated microfluidic devices for bacterial DNA purification without chaotropic solutions.

We present a disposable polymeric microfluidic device capable of reversibly binding and purifying Salmonella DNA through solid phase extraction (SPE). The microfluidic channels are first oxygen plasma treated and simultaneously micro-nanotextured, and then functionalized with amine groups via modification with L-histidine or poly-L-histidine. L-Histidine and poly-L-histidine bind on the plasma treated chip surface, and are not detached when rinsing with DNA purification protocol buffers. A pH-dependent protocol is applied on-chip to purify Salmonella DNA, which is first bound on the protonated amines at a pH (5.0) lower than their pKa of surface amine-groups which is 6.0 and then released at a pH higher than the pKa value (10.5). It was found that modification with poly-L-histidine resulted in higher surface density of amine groups onto microfluidic channel. Using the chip modified with poly-L-histidine, high recovery efficiency of at least 550 ng of isolated Salmonella DNA as well as DNA purification from Salmonella cell lysates corresponding to less than 5000 cells or 0.026 ng of Salmonella DNA was achieved. The protocol developed does not require ethanol or chaotropic solutions typically used in DNA purification, which are known inhibitors for downstream operations such as polymerase chain reactions (PCR) and which can also attack some polymeric microfluidic materials. Therefore, the microfluidic device and the related protocol hold promise for facile incorporation in microfluidics and Lab-on-a-chip (LOC) platforms for pathogen detection or in general for DNA purification.

Plasma micro-nanotextured polymeric micromixer for DNA purification with high efficiency and dynamic range.

We present a polymeric microfluidic chip capable of purifying DNA through solid phase extraction. It is designed to be used as a module of an integrated Lab-on-chip platform for pathogen detection, but it can also be used as a stand-alone device. The microfluidic channels are oxygen plasma micro-nanotextured, i.e. randomly roughened in the micro-nano scale, a process creating high surface area as well as high density of carboxyl groups (COOH). The COOH groups together with a buffer that contains polyethylene glycol (PEG), NaCl and ethanol are able to bind DNA on the microchannel surface. The chip design incorporates a mixer so that sample and buffer can be efficiently mixed on chip under continuous flow. DNA is subsequently eluted in water. The chip is able to isolate DNA with high recovery efficiency (96± 11%) in an extremely large dynamic range of prepurified Salmonella DNA as well as from Salmonella cell lysates that correspond to a range of 5 to 1.9 × 108 cells (0.263 fg to 2 × 500 ng). The chip was evaluated via absorbance measurements, polymerase chain reaction (PCR), and gel electrophoresis.